RIG-I-like Receptors Signaling Research Articles

Cooperative effects of RIG-I-like receptor signaling and IRF1 on DNA damage-induced cell death

Properly responding to DNA damage is vital for eukaryotic cells, including the induction of DNA repair, growth arrest and, as a last resort to prevent neoplastic transformation, cell death. Besides being crucial for ensuring homeostasis, the same pathways and mechanisms are at the basis of chemoradiotherapy in cancer treatment, which involves therapeutic induction of DNA damage by chemical or physical (radiological) measures. Apart from typical DNA damage response mediators, the relevance of cell-intrinsic antiviral signaling pathways in response to DNA breaks has recently emerged. Originally known for combatting viruses via expression of antiviral factors including interferons (IFNs) and establishing of an antiviral state, RIG-I-like receptors (RLRs) were found to be critical for adequate induction of cell death upon the introduction of DNA double-strand breaks. We here show that presence of IRF3 is crucial in this process, most likely through direct activation of pro-apoptotic factors rather than transcriptional induction of canonical downstream components, such as IFNs. Investigating genes reported to be involved in both DNA damage response and antiviral signaling, we demonstrate that IRF1 is an obligatory factor for DNA damage-induced cell death. Interestingly, its regulation does not require activation of RLR signaling, but rather sensing of DNA double-strand breaks by ATM and ATR. Hence, even though independently regulated, both RLR signaling and IRF1 are essential for full-fledged induction/execution of DNA damage-mediated cell death programs. Our results not only support more broadly developing IRF1 as a biomarker predictive for the effectiveness of chemoradiotherapy, but also suggest investigating a combined pharmacological stimulation of RLR and IRF1 signaling as a potential adjuvant regimen in tumor therapy.

IRF4b and IRF8 Negatively Regulate RLR-Mediated NF-κB Signaling by Targeting MITA for Degradation in Teleost Fish.

Mediator of IRF3 activation (MITA) is a significant signal adaptor in the retinoic acid-inducible gene-I like receptor (RLR) signaling pathway and plays an important role in the innate immune system. As a transcription factor, nuclear factor kappa B (NF-κB) can be available in many signaling pathways including the RLR signaling pathway and relative to biological processes like immune responses. In this study, it is determined that IRF4b and IRF8 can have a negative effect on NF-κB signaling pathway mediated by MITA in fish. Firstly, it is found that IRF4b and IRF8 have an inhibitory function on MITA-mediated NF-κB signaling pathway. It is interesting that IRF4b and IRF8 have similar functions to achieve precise downregulated and the degradation of MITA through the ubiquitin-proteasome pathway. IRF is taken as the core domain of IRF4b or IRF8 for the downregulation to MITA. This study provides data on MITA-mediated NF-κB signaling pathway in teleost fish and provides new insights into the regulatory mechanism in fish immune system.

Grass Carp Reovirus Nonstructural Proteins Avoid Host Antiviral Immune Response by Targeting the RLR Signaling Pathway.

Grass carp reovirus (GCRV) is a highly virulent RNA virus that mainly infects grass carp and causes hemorrhagic disease. The roles of nonstructural proteins NS38 and NS80 of GCRV-873 in the viral replication cycle and viral inclusion bodies have been established. However, the strategies that NS38 and NS80 used to avoid host antiviral immune response are still unknown. In this study, we report the negative regulations of NS38 and NS80 on the RIG-I-like receptors (RLRs) antiviral signaling pathway and the production of IFNs and IFN-stimulated genes. First, both in the case of overexpression and GCRV infection, NS38 and NS80 inhibited the IFN promoter activation induced by RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7 and mRNA abundance of key antiviral genes involved in the RLR-mediated signaling. Second, both in the case of overexpression and GCRV infection, NS38 interacted with piscine TBK1 and IRF3, but not with piscine RIG-I, MDA5, MAVS, and TNF receptor-associated factor (TRAF) 3. Whereas NS80 interacted with piscine MAVS, TRAF3, and TBK1, but not with piscine RIG-I, MDA5, and IRF3. Finally, both in the case of overexpression and GCRV infection, NS38 inhibited the formation of the TBK1-IRF3 complex, but NS80 inhibited the formation of the TBK1-TRAF3 complex. Most importantly, NS38 and NS80 could hijack piscine TBK1 and IRF3 into the cytoplasmic viral inclusion bodies and inhibit the translocation of IRF3 into the nucleus. Collectively, all of these data demonstrate that GCRV nonstructural proteins can avoid host antiviral immune response by targeting the RLR signaling pathway, which prevents IFN-stimulated gene production and facilitates GCRV replication.

Amantadine-assembled nanostimulator enhances dimeric RBD antigen-elicited cross-neutralization against SARS-CoV-2 strains

There is an urgent need to develop new vaccination strategies to elevate the cross-neutralization against different SARS-CoV-2 strains. In this study, we construct the spherical amantadine-assembled nanostimulator (AAS). Amantadine as immunostimulating molecules are displayed on the outermost layer of AAS. Molecular mechanism analysis reveals that AAS can activate RIG-I-like receptor (RLR) signaling pathway to increase the expression of type I interferons in vivo. AAS-mediated activation of RLR signaling pathway further promotes the maturation and proliferation of dendritic cells (DCs) and T helper cells (Ths), finally activating B cells to produce potent antibody responses. In performance evaluation experiments, the mixture of AAS and dimeric RBD significantly enhances RBD-specific humoral responses (4-fold IgG, 3.5-fold IgG2a, 3.3-fold IgG2b, 3.8-fold IgG3 and 1.3-fold IgM), in comparison to aluminum adjuvant-assistant dimeric RBD. Importantly, AAS dramatically elevates dimeric RBD-elicited cross-neutralization against different SARS-CoV-2 strains such as Wuhan-Hu-1 (9-fold), B.1.1.7 (UK variant, 15-fold), B.1.351 (South African variant, 4-fold) and B.1.617.2 (India variant, 7-fold). Our study verifies the mechanism of AAS in activating RLR signaling pathway in host immune system and highlights the power of AAS in improving antigen-elicited cross-neutralization against different SARS-CoV-2 strains.

PolyI:C attenuates transforming growth factor-β signaling to induce cytostasis of surrounding cells by secreted factors in triple-negative breast cancer.

The activation of RIG‐I‐like receptor (RLR) signaling in cancer cells is widely recognized as a critical cancer therapy method. The expected mechanism of RLR ligand‐mediated cancer therapy involves the promotion of cancer cell death and strong induction of interferon (IFN)‐β that affects the tumor microenvironment. We have recently shown that activation of RLR signaling in triple‐negative breast cancer cells (TNBC) attenuates transforming growth factor‐β (TGF‐β) signaling, which partly contributes to the promotion of cancer cell pyroptosis. However, the consequences of suppression of TGF‐β signaling by RLR ligands with respect to IFN‐β‐mediated tumor suppression are not well characterized. This study showed that transfection of a typical RLR ligand polyI:C in cancer cells produces significant levels of IFN‐β, which inhibits the growth of the surrounding cancer cells. In addition, IFN‐β‐induced cell cycle arrest in surrounding cancer cells was inhibited by the expression of constitutively active Smad3. Constitutively active Smad3 suppresses IFN‐β expression through the alleviation of IFN regulatory factor 3 binding to the canonical target genes, as suggested by ChIP sequencing analysis. Based on these findings, a new facet of the protumorigenic function of TGF‐β that suppresses IFN‐β expression is suggested when RLR‐mediated cancer treatment is used in TNBC.

Activation and Evasion of RLR Signaling by DNA Virus Infection.

Antiviral innate immune response triggered by nucleic acid recognition plays an extremely important role in controlling viral infections. The initiation of antiviral immune response against RNA viruses through ligand recognition of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) was extensively studied. RLR’s role in DNA virus infection, which is less known, is increasing attention. Here, we review the research progress of the ligand recognition of RLRs during the DNA virus infection process and the viral evasion mechanism from host immune responses.

The Kinase MAP4K1 Inhibits Cytosolic RNA-Induced Antiviral Signaling by Promoting Proteasomal Degradation of TBK1/IKKε.

ABSTRACTTANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) mediates robust production of type I interferons (IFN-I) and proinflammatory cytokines in response to acute viral infection. However, excessive or prolonged production of IFN-I is harmful and even fatal to the host by causing autoimmune disorders. In this study, we identified mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) as a negative regulator in the RIG-I-like receptor (RLR) signaling pathway. MAP4K1, a member of Ste20-like serine/threonine kinases, was previously known as a prominent regulator in adaptive immunity by downregulating T-cell receptor (TCR) signaling and B-cell receptor (BCR) signaling. However, its role in regulating antiviral innate immune signaling is still unclear. This study reports an undiscovered role of MAP4K1, which inhibits RLR signaling by targeting TBK1/IKKε for proteasomal degradation via the ubiquitin ligase DTX4. We initially identify MAP4K1 as an interacting partner of TBK1 by yeast two-hybrid screens and subsequently investigate its function in RLR-mediated antiviral signaling pathways. Overexpression of MAP4K1 significantly inhibits RNA virus-triggered activation of IFN-β and the production of proinflammatory cytokines. Consistently, knockdown or knockout experiments show opposite effects. Furthermore, MAP4K1 promotes the degradation of TBK1/IKKε by K48-linked ubiquitination via DTX4. Knockdown of DTX4 abrogated the ubiquitination and degradation of TBK1/IKKε. Collectively, our results identify that MAP4K1 acts as a negative regulator in antiviral innate immunity by targeting TBK1/IKKε, discover a novel TBK1 inhibitor, and extend a novel functional role of MAP4K1 in immunity.IMPORTANCE TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε) mediates robust production of type I interferons (IFN-I) and proinflammatory cytokines to restrict the spread of invading viruses. However, excessive or prolonged production of IFN-I is harmful to the host by causing autoimmune disorders. In this study, we identified that mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1) is a negative regulator in the RLR signaling pathway. Notably, MAP4K1 promotes the degradation of TBK1/IKKε by K48-linked ubiquitination via the ubiquitin ligase DTX4, leading to the negative regulation of the IFN signaling pathway. Previous studies showed that MAP4K1 has a pivotal function in adaptive immune responses. This study identifies that MAP4K1 also plays a vital role in innate immunity and outlines a novel mechanism by which the IFN signaling pathway is tightly controlled to avoid excessive inflammation. Our study documents a novel TBK1 inhibitor, which serves as a potential therapeutic target for autoimmune diseases, and elucidated a significant function for MAP4K1 linked to innate immunity in addition to subsequent adaptive immunity.

N4BP3 Regulates RIG-I-Like Receptor Antiviral Signaling Positively by Targeting Mitochondrial Antiviral Signaling Protein.

Mitochondrial antiviral signaling protein (MAVS), an adaptor protein, is activated by RIG-I, which is critical for an effective innate immune response to infection by various RNA viruses. Viral infection causes the RIG-I-like receptor (RLR) to recognize pathogen-derived dsRNA and then becomes activated to promote prion-like aggregation and activation of MAVS. Subsequently, through the recruitment of TRAF proteins, MAVS activates two signaling pathways mediated by TBK1-IRF3 and IKK- NF-κb, respectively, and turns on type I interferon and proinflammatory cytokines. This study discovered that NEDD4 binding protein 3 (N4BP3) is a positive regulator of the RLR signaling pathway by targeting MAVS. Overexpression of N4BP3 promoted virus-induced activation of the interferon-β (IFN-β) promoter and interferon-stimulated response element (ISRE). Further experiments showed that knockdown or knockout N4BP3 impaired RIG-I-like receptor (RLR)-mediated innate immune response, induction of downstream antiviral genes, and cellular antiviral responses. We also detected that N4BP3 could accelerate the interaction between MAVS and TRAF2. Related experiments revealed that N4BP3 could facilitate the ubiquitination modification of MAVS. These findings suggest that N4BP3 is a critical component of the RIG-I-like receptor (RLR)-mediated innate immune response by targeting MAVS, which also provided insight into the mechanisms of innate antiviral responses.

Robust innate immune responses at the placenta during early gestation may limit in utero HIV transmission.

In 2019, >90% of new HIV infections in infants globally occurred vertically. Studies suggest intrauterine transmission most often occurs in the third trimester; however, there are no mechanistic studies to support these observations. We therefore obtained early/mid-gestation and term placentae from 20 HIV/Hepatitis B/CMV negative women. Isolated primary placental macrophages (Hofbauer cells [HCs]) were exposed to HIV-1BaL and/or interferon (IFN)-α, IFN-β, IFN-λ1, and RIG-I-like receptor (RLR) agonists. qRT-PCR, FACS, ELISA, Luminex, and Western blot analyses determined expression of activation markers, co-receptors, viral antigen, cytokines, antiviral genes, and host proteins. Early gestation HCs express higher levels of CCR5 and exhibit a more activated phenotype. Despite downregulation of CCR5, term HCs were more susceptible to HIV replication. Early gestation HCs displayed a more activated phenotype than term HCs and HIV exposure lead to the further up-regulation of T-cell co-stimulatory and MHC molecules. Limited HIV replication in early/mid gestation HCs was associated with increased secretion of anti-inflammatory cytokines, chemokines, and a more robust antiviral immune response. In contrast, term HCs were more susceptible to HIV replication, associated with dampening of IFN-induced STAT1 and STAT2 protein activation. Treatment of early/mid gestation and term HCs, with type I IFNs or RLR agonists reduced HIV replication, underscoring the importance of IFN and RLR signaling in inducing an antiviral state. Viral recognition and antiviral immunity in early gestation HCs may prevent in utero HIV infection, whereas diminished antiviral responses at term can facilitate transmission. Defining mechanisms and specific timing of vertical transmission are critical for the development of specific vaccines and antiviral therapeutics to prevent new HIV infections in children globally.

Viperin interacts with PEX19 to mediate peroxisomal augmentation of the innate antiviral response.

Peroxisomes are recognized as significant platforms for the activation of antiviral innate immunity where stimulation of the key adapter molecule mitochondrial antiviral signaling protein (MAVS) within the RIG-I like receptor (RLR) pathway culminates in the up-regulation of hundreds of ISGs, some of which drive augmentation of multiple innate sensing pathways. However, whether ISGs can augment peroxisome-driven RLR signaling is currently unknown. Using a proteomics-based screening approach, we identified Pex19 as a binding partner of the ISG viperin. Viperin colocalized with numerous peroxisomal proteins and its interaction with Pex19 was in close association with lipid droplets, another emerging innate signaling platform. Augmentation of the RLR pathway by viperin was lost when Pex19 expression was reduced. Expression of organelle-specific MAVS demonstrated that viperin requires both mitochondria and peroxisome MAVS for optimal induction of IFN-β. These results suggest that viperin is required to enhance the antiviral cellular response with a possible role to position the peroxisome at the mitochondrial/MAM MAVS signaling synapse, furthering our understanding of the importance of multiple organelles driving the innate immune response against viral infection.

Targeting the RIG-I-like receptor signaling pathway to improve the efficacy of immunotherapy in mucosal and uveal melanoma.

e21593 Background: Patients with cutaneous (CM) or acral (AM) melanoma have high response rates to immune checkpoint blockade (ICB) therapies including anti-CTLA4, anti-PD1, and their combination. In contrast, patients with mucosal (MM) or uveal (UM) melanoma have low ICB response rates and a poorer overall prognosis. In this study, we analyzed melanoma transcriptomes to identify potential mechanisms underlying ICB resistance in MM and UM. We also evaluated epigenetic modifying drugs as potential agents to improve ICB responses in these patients. Methods: RNA sequencing was performed on 13 MM, 8 AM, and 24 CM patient-derived xenograft tumors. These data were analyzed along with published RNA sequencing data for 80 UM and 443 CM tumors. MM and UM cells were treated with several different classes of epigenetic modifying drugs, and the ICB-resistant YUMM1.7 mouse melanoma syngeneic model was used to evaluate the tumor growth effect of one particular drug, decitabine, at low (2.5 ug) and intermediate (10 ug) doses in combination with anti-PD1 (200 ug). Results: We identified 2513 differentially expressed genes (DEG) between MM and CM tumors (ANODEV adjusted p-value < 0.05, FDR < 0.1) compared to only 495 DEG between AM and CM. Ingenuity Pathway Analysis (IPA) analysis of the 2513 DEG in MM identified the RIG-I-like receptor (RLR) pathway as inhibited in MM versus CM ( p < 1x10-5). The RLR pathway was also inhibited in ICB-resistant CM versus ICI-sensitive (anti-CTLA4, p < 0.006 and anti-PD1, p < 0.0002) and in UM versus CM ( p < 1x10-8), but was not inhibited in AM. The RLR pathway is an innate immune pathogen sensing pathway that is critical for ICB responses in CM; therefore, we sought to identify FDA-approved drugs which activate the RLR pathway in MM and UM. Since RLR signaling is frequently repressed by epigenetic silencing, we screened direct DNMT1 inhibitors 5’-azacitidine (AZA) and 5’-aza-deoxycitidine (decitabine, DEC), several indirect DNMT1 inhibitors, and two HDAC inhibitors. We found that only DEC strongly induced the expression of RLR pathway genes in MM, UM, and ICB-resistant CM cells. Compared to vehicle, treatment of YUMM1.7 tumors with single agent anti-PD1 ( p > 0.87) and low dose ( p > 0.90) or intermediate dose ( p > 0.80) DEC had no effect on tumor growth. However, the addition of either low or intermediate dose DEC to anti-PD1 showed a trend of decreased tumor growth (low dose, 31% decrease, p = 0.22 and intermediate dose, 41% decrease, p = 0.55). Conclusions: We identified low RLR pathway signaling as one possible mechanism underlying ICB resistance in MM and UM. We show that an FDA-approved DNMT1 inhibitor, decitabine, strongly induces RLR pathway genes in MM and UM cells in vitro and leads to tumor growth suppression in combination with anti-PD1 in vivo. Therefore, co-administration of decitabine and anti-PD1 agents is a rational and novel strategy for improving outcomes for MM and UM patients.

RNF39 mediates K48-linked ubiquitination of DDX3X and inhibits RLR-dependent antiviral immunity.

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) are major cytosolic RNA sensors and play crucial roles in initiating antiviral innate immunity. Furthermore, RLRs have been implicated in multiple autoimmune disorders. Thus, RLR activation should be tightly controlled to avoid detrimental effects. "DEAD-box RNA helicase 3, X-linked" (DDX3X) is a key adaptor in RLR signaling, but its regulatory mechanisms remain unknown. Here, we show that the E3 ubiquitin ligase RNF39 inhibits RLR pathways through mediating K48-linked ubiquitination and proteasomal degradation of DDX3X. Concordantly, Rnf39 deficiency enhances RNA virus-triggered innate immune responses and attenuates viral replication. Thus, our results uncover a previously unknown mechanism for the control of DDX3X activity and suggest RNF39 as a priming intervention target for diseases caused by aberrant RLR activation.

The negative regulation of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling pathway in fish

At each stage of innate immune response, there are stimulatory and inhibitory signals that modulate the strength and character of the response. RIG-I-like receptor (RLR) signaling pathway plays pivotal roles in antiviral innate immune response. Recent studies have revealed the molecular mechanisms that viral infection leads to the activation of RLRs-mediated downstream signaling cascades and the production of type I interferons (IFNs). However, antiviral immune responses must be tightly regulated in order to prevent detrimental type I IFNs production. Previous reviews have highlighted negative regulation of RLR signaling pathway, which mainly target to directly regulate RIG-I, MDA5, MAVS and TBK1 function in mammals. In this review, we summarize recent advances in our understanding of negative regulators of RLR signaling pathway in teleost, with specific focus on piscine and viral regulatory mechanisms that directly or indirectly inhibit the function of RIG-I, MDA5, LGP2, MAVS, TRAF3, TBK1, IRF3 and IRF7 both in the steady state or upon viral infection. We also further discuss important directions for future studies, especially for non-coding RNAs and post-translational modifications via fish specific TRIM proteins. The knowledge of negative regulators of RLR signaling pathway in teleost will shed new light on the critical information for potential therapeutic purposes.

Viral Evasion of RIG-I-Like Receptor-Mediated Immunity through Dysregulation of Ubiquitination and ISGylation.

Viral dysregulation or suppression of innate immune responses is a key determinant of virus-induced pathogenesis. Important sensors for the detection of virus infection are the RIG-I-like receptors (RLRs), which, in turn, are antagonized by many RNA viruses and DNA viruses. Among the different escape strategies are viral mechanisms to dysregulate the post-translational modifications (PTMs) that play pivotal roles in RLR regulation. In this review, we present the current knowledge of immune evasion by viral pathogens that manipulate ubiquitin- or ISG15-dependent mechanisms of RLR activation. Key viral strategies to evade RLR signaling include direct targeting of ubiquitin E3 ligases, active deubiquitination using viral deubiquitinating enzymes (DUBs), and the upregulation of cellular DUBs that regulate RLR signaling. Additionally, we summarize emerging new evidence that shows that enzymes of certain coronaviruses such as SARS-CoV-2, the causative agent of the current COVID-19 pandemic, actively deISGylate key molecules in the RLR pathway to escape type I interferon (IFN)-mediated antiviral responses. Finally, we discuss the possibility of targeting virally-encoded proteins that manipulate ubiquitin- or ISG15-mediated innate immune responses for the development of new antivirals and vaccines.

Innate Immune Sensing of Viruses and Its Consequences for the Central Nervous System.

Viral infections remain a global public health concern and cause a severe societal and economic burden. At the organismal level, the innate immune system is essential for the detection of viruses and constitutes the first line of defense. Viral components are sensed by host pattern recognition receptors (PRRs). PRRs can be further classified based on their localization into Toll-like receptors (TLRs), C-type lectin receptors (CLR), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), NOD-like receptors (NLRs) and cytosolic DNA sensors (CDS). TLR and RLR signaling results in production of type I interferons (IFNα and -β) and pro-inflammatory cytokines in a cell-specific manner, whereas NLR signaling leads to the production of interleukin-1 family proteins. On the other hand, CLRs are capable of sensing glycans present in viral pathogens, which can induce phagocytic, endocytic, antimicrobial, and pro- inflammatory responses. Peripheral immune sensing of viruses and the ensuing cytokine response can significantly affect the central nervous system (CNS). But viruses can also directly enter the CNS via a multitude of routes, such as the nasal epithelium, along nerve fibers connecting to the periphery and as cargo of infiltrating infected cells passing through the blood brain barrier, triggering innate immune sensing and cytokine responses directly in the CNS. Here, we review mechanisms of viral immune sensing and currently recognized consequences for the CNS of innate immune responses to viruses.

Therapeutic potency and mechanisms of polyI:C in triple negative breast cancer

Triple negative breast cancer (TNBC) is one of the most aggressive types of cancers that requires more innovative therapy. Activating retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) including melanoma differentiation-associated gene 5 (MDA5) and RIG-I in cancer cells such as by transfection of dsRNA (e.g. polyI:C) is suggested as new beneficial strategy to combat cancer cells by triggering cancer cell death including TNBC. However, crosstalk between RLR signaling pathway and other tumor promoting signaling pathways is not fully elucidated. Here we showed that cytosolic induction of polyI:C which is now in clinical trials by intratumoral transfection suppressed tumor-promoting transforming growth factor-β (TGF-β) signaling in TNBC by attenuating phosphorylation of Smad3. Abrogation of TGF-β signaling by transfection of polyI:C accelerated cell death, which was inhibited by forced expression of constitutively active Smad3. We further characterized cell death and found polyI:C transfection caused pyroptosis accompanied by GSDME cleavage. Intratumoral polyI:C treatment in vivo resulted in suppression of tumor progression with attenuation of pSmad3. These results suggested that transfection of polyI:C is a promising method to combat TNBC through attenuation of TGF-β signaling.

Attenuation of Antiviral Immune Response Caused by Perturbation of TRIM25-Mediated RIG-I Activation under Simulated Microgravity

Microgravity is a major environmental factor of space flight that triggers dysregulation of the immune system and increases clinical risks for deep-space-exploration crews. However, systematic studies and molecular mechanisms of the adverse effects of microgravity on the immune system in animal models are limited. Here, we establish a ground-based zebrafish disease model of microgravity for the research of space immunology. RNA sequencing analysis demonstrates that the retinoic-acid-inducible gene (RIG)-I-like receptor (RLR) and the Toll-like receptor (TLR) signaling pathways are significantly compromised by simulated microgravity (Sμg). TRIM25, an essential E3 for RLR signaling, is inhibited under Sμg, hampering the K63-linked ubiquitination of RIG-I and the following function-induction positive feedback loop of antiviral immune response. These mechanisms provide insights into better understanding of the effects and principles of microgravity on host antiviral immunity and present broad potential implications for developing strategies that can prevent and control viral diseases during space flight.

Transcriptome analysis reveals temperature-dependent early immune response in flounder (Paralichthys olivaceus) after Hirame novirhabdovirus (HIRRV) infection

Hirame novirhabdovirus (HIRRV), as a highly pathogenic fish virus, is frequently prevalent in a variety of aquaculture fish in recent years, which seriously threatens the healthy development of aquaculture industry. Epidemiological studies show that the outbreak of HIRRV is obviously temperature dependent. Virus proliferation experiments in vitro and in vivo at different temperatures indicate the antiviral response of flounder is a main reason affect the replication of HIRRV. The RNA-Seq was used to analyze the different antiviral response in flounder which infected with HIRRV at different temperatures, the experiment set two temperatures of 10 °C and 20 °C. The flounder infected with HIRRV by artificial injection, and the spleens were collected at 24 h after infection. Meanwhile, the fish injected with EPC supernatant at different temperatures were set as control groups. It can obtain four pairwise comparison groups if determine a single variable, and the differentially expressed genes were further selected. The results showed that after infection with HIRRV at 10 °C and 20 °C, the differentially expressed genes in spleen were significantly enriched in inflammatory and immune-related pathways like Arachidonic acid metabolism, Cytokine-cytokine receptor interaction, Toll-like receptor (TLR) signaling pathway, RIG-I-like receptor (RLRs) signaling pathway, NOD-like receptor (NLR) signaling pathway and Cytosolic DNA-sensing pathway etc. In addition, the expression of phagocytes, lysosomes, endocytosis related genes were significantly upregulated at high temperature whether HIRRV positive or not. But compared to the infected flounder at 10 °C, some genes of RLRs signaling pathway were significantly upregulated at 20 °C, it can be speculated that RLRs pathway may be related to the anti-HIRRV response of flounder. Therefore, key genes of RLRs signaling pathway including mda5, lgp2, mita, mavs, irf3, irf7, ifn I-3 and ifn-γ were selected, and the temporal expression patterns of these genes in infected flounder at different temperatures were further detected by qRT-PCR. The results showed that HIRRV infection can significantly stimulate and activate the RLRs pathway of flounder, and the response level of this pathway was significantly higher at 20 °C than 10 °C. In general, this study provides important data for the further study about the pathogenesis of HIRRV infection in flounder.

The p150 Isoform of ADAR1 Blocks Sustained RLR signaling and Apoptosis during Influenza Virus Infection.

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-β expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-β expression and apoptosis during viral infection.

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression.

The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/β) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (Lpro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/β gene transcription; however, the exact mechanism is unknown. The proteolytic activity of Lpro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, Lpro has been demonstrated to have deubiquitination/deISGylation activity. Lpro’s deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/β gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by Lpro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing Lpro. In vitro cleavage experiments revealed that Lpro cleaves TBK1 at residues 692–694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-Lpro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK αVβ6 cells. We set out to dissect Lpro’s ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity to determine their relative contributions to the reduction of IFN-α/β gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of Lpro in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of Lpro. Characterization of the effects of these mutations revealed that Lpro’s ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-β gene transcription.